Using a cohort of more than 550 subjects with a wide variety of eosinophilic disorders, ranging from benign eosinophilia to eosinophilic leukemia, we have continued to identify and characterize novel subgroups of patients with eosinophilia. In this regard, we continue to explore surface receptor expression on eosinophils, measures of eosinophil activation and responses to targeted therapies as ways to increase our understanding of the underlying pathophysiologies in diverse groups of patients with HES. In anticipation of a planned clinical trial with a monoclonal antibody to the inhibitory receptor, Siglec-8, in patients with eosinophilic gastritis, we characterized surface and soluble Siglec-8 levels in normal (ND) and eosinophilic (EO) subjects and assessed the efficacy of anti-Siglec-8 antibodies in inducing eosinophil cell death in vitro. Siglec-8 was consistently expressed on eosinophils from ND and EO and did not correlate with absolute eosinophil count (AEC) or disease activity. Soluble Siglec-8 levels were measurable in serum from most donors, unrelated to AEC or Siglec-8 surface expression. Monoclonal chimeric antibodies to Siglec-8 IgG1 and IgG4 were equally effective at inducing eosinophil cell death after overnight IL-5 priming. In contrast, killing of purified eosinophils without IL-5 was only seen in EO subjects, and NK-mediated eosinophil killing was seen only with the afucosylated anti-Siglec-8 IgG1. Finally, treatment of humanized mice with anti-Siglec antibody led to robust depletion of IL-5-induced eosinophilia in vivo. These data support the use of anti-Siglec-8 antibodies as potential therapeutics for patients with eosinophilic disorders (Legrand et al. J Allergy Clin Immunol, 2019). In a separate study, we demonstrated that the inhibitory receptor, Siglec-7 was constitutively expressed on blood eosinophils from eosinophilic and normal individuals and that surface expression was correlated with absolute eosinophil count (AEC). Siglec-7 was upregulated on purified eosinophils after in vitro stimulation with GM-CSF or IL-5. Serum sSiglec-7 was detectable in 133/144 subjects tested and correlated with AEC. Siglec-7 cross-linking downmodulated eosinophil activation, as evidenced by inhibition of GM-CSF-induced release of eosinophil granule proteins and inflammatory mediators but did not promote eosinophil apoptosis. Targeting of Siglec-7 on eosinophils might enhance treatment efficacy in eosinophil-driven disorders. Conversely, therapeutic interventions that inhibit Siglec-7 in other conditions could have unanticipated consequences and promote eosinophilic inflammation. Finally, we have continued to study the utility of eosinophil granule protein (EGP) measurement in the blood, urine and other body fluids as a biomarker for eosinophil activation (Makiya et al. J Immunol Methods, 2014) with recent data demonstrating the utility of urine EGP levels in the context of a phase 3 trial of mepolizumab for EGPA (Khoury et al. J Allergy Clin Immunol 2019; 143(2): AB288). Therapy for eosinophilic disorders remains a primary focus of our group. In the past year, we completed a placebo-controlled, double-blind phase 2 trial of benralizumab in HES (MedImmune/Astra Zeneca) (Kuang et al. N Engl J Med 2019). Benralizumab is an afucosylated antibody to IL-5 receptor alpha that has shown clinical efficacy in patients with eosinophilic asthma. The current trial stemmed, in part, from our prior pre-clinical work examining IL-5 receptor levels in patients with eosinophilia and/or mastocytosis (Wilson et al. J Allergy Clin Immunol 2011). Twenty adults with PDGFRA-negative HES and AEC 1000 cells/mm3 on stable background therapy were enrolled between April 2014 and January 2017 and randomized to receive benralizumab or placebo monthly for 3 months. while on stable background therapy. At week 12, subjects received open-label benralizumab, and AEC was unblinded beginning at week 13. Depending on the results of the week 13 AEC, subjects were eligible to receive monthly benralizumab with tapering of background therapy. The study met the primary endpoint with a >50% reduction in AEC in 9/10 subjects who received benralizumab compared to 3/10 who received placebo. The most common drug-related adverse events, headache and an elevated lactate dehydrogenase level, occurred in 32% of the patients after the first dose of benralizumab and resolved within 48 hours in all patients. Other adverse events occurred with similar frequency in the two groups. Biopsies of affected tissues were performed in 7 subjects and showed resolution of tissue eosinophilia at week 24 (Kuang et al. J Allergy Clin Immunol 2018; 141(2): AB196). During the open-label phase, clinical and hematologic responses were observed in 17 of 19 patients (89%) and were sustained for 48 weeks in 14 of 19 patients (74%). In addition to the benralizumab trial, we participated in a recently completed multicenter trial of AK002 (an afucosylated antibody to Siglec-8) for the treatment of eosinophilic gastritis (analysis ongoing) and continue to follow patients on enrolled on investigator-initiated trials of benralizumab and dexpramipexole, as well as those receiving compassionate use mepolizumab. Long-term followup of patients on these studies has provided insight into the effects of eosinophil depletion in humans and the mechanisms driving eosinophilia and eosinophil activation in HES. We also continue to explore standard therapies for HES. Glucocorticoids (GC) are the mainstay of therapy for a variety of eosinophil-associated disorders, including hypereosinophilic syndromes (HES), although responses are not universal and GC are associated with significant toxicity. Prior to the first therapeutic use of GC, it was observed that the administration of exogenous adrenocorticotropic hormone or hydrocortisone led to a rapid, profound, and transient eosinopenia in humans. Although this is likely an important first step in the clinical response to GC, little is known about the underlying mechanism. To address this, we assessed the global transcriptional response to GC in human eosinophils through RNA sequencing of peripheral blood eosinophils following a single dose of oral prednisone (1 mg/kg) in three healthy subjects with asymptomatic hypereosinophilia. The kinetics of GC-induced eosinopenia was consistent across the three subjects, with the initial decline occurring between 60 and 120 minutes after GC administration. Among the 414 genes differentially expressed in the first hour after GC administration, the most common were genes associated with apoptosis and CXCR4. Upregulation of CXCR4 protein expression on eosinophils was confirmed in vitro and in vivo, suggesting that this surface receptor is key in the early egress of eosinophils from the circulation (Khoury et al. Allergy 2018). Pursuant to these studies, we have examined the eosinophil response to GC in a rhesus monkey model using Zirconium-labelled eosinophils. Data using this model demonstrates early trafficking of eosinophils to the bone marrow that is blocked by pre-treatment with plerixafor (a CXCR4 blocking antibody) (Legrand, Hong et al. JACI 143(2):AB583). Finally, we continue to explore mechanisms of GC resistance in our cohort of patients with HES. Clinical data and samples from 26 patients with HES enrolled on a prospective study of GC-responsiveness and 23 patients with HES enrolled on a natural history study of eosinophilia for whom response to GC was known were analyzed retrospectively (Stokes et al. Clin Exp Allergy 2019, in revision). Our data suggest that the mechanism of GC resistance in HES is not due to a global phenomenon affecting all lineages, but may be due, at least in some patients, to impairment of eosinophil apoptosis by increased levels of IL-5.